KR20120123445A - Pharmaceutical composition for treating a metabolic syndrome - Google Patents

Abstract

The present invention provides at least one FGF-21 (fibroblast growth factor 21) compound, at least one for the treatment of at least one metabolic syndrome and / or atherosclerosis, in particular diabetes, dyslipidemia, obesity and / or hyperlipidemia A pharmaceutical composition containing a GLP-1R (glucagon-like peptide-1 receptor) agonist and optionally at least one anti-diabetic drug and / or at least one DPP-4 (dipeptidyl peptidase-4) inhibitor. .

Description

PHARMACEUTICAL COMPOSITION FOR TREATING A METABOLIC SYNDROME

The present invention provides at least one FGF-21 (fibroblast growth factor 21) compound, at least one for the treatment of at least one metabolic syndrome and / or atherosclerosis, in particular diabetes, dyslipidemia, obesity and / or hyperlipidemia A pharmaceutical composition containing a GLP-1R (glucagon-like peptide-1 receptor) agonist and optionally at least one anti-diabetic drug and / or at least one DPP-4 (dipeptidyl peptidase-4) inhibitor. .

Diabetic diabetes is characterized by its clinical manifestations, namely non-insulin-dependent or adult developmental forms, also known as type 2 diabetes, and insulin-dependent or adolescent developmental forms, also known as type 1 diabetes. Signs of clinical symptoms of type 2 diabetes and underlying obesity usually appear at ages 40 and older. In contrast, type 1 diabetes usually indicates rapid development of the disease, often before age 30. The disease is a metabolic disorder in humans with a prevalence of approximately 1% in the general population, one quarter of which is type 1 and three quarters of which is type 2 diabetes. Type 2 diabetes is a disease characterized by high-blood circulating blood sugar, insulin and corticosteroid levels.

At present, various pharmacological approaches exist for the treatment of type 2 diabetes, which can be used individually or in combination and act through different modes of action:

1) sulfonylureas stimulate insulin secretion;

2) biguanides (metformin) act by promoting glucose use, reducing glucose production in the liver, and reducing intestinal glucose production;

3) oc-glucosidase inhibitors (acarbose, miglitol) reduce the post-prandial hyperglycemia by slowing digestion of carbohydrates and consequently delaying absorption from the intestine;

4) Thiazolidinedione (troglitazone) promotes glucose use in peripheral tissues by enhancing insulin action;

5) Insulin stimulates tissue glucose use and inhibits glucose production in the liver.

However, most of the agents have limited efficacy and do not solve the most important problem, reduced β-cell function and associated obesity. Obesity is a chronic disease that is quite overwhelming in modern society and is associated with a number of medical problems, including diabetes mellitus, insulin resistance, hypertension, hypercholesterolemia and coronary heart disease. It also has a significant correlation with diabetes and insulin resistance, the latter of which is usually accompanied by hyperinsulinemia or hyperglycemia or both. Type 2 diabetes is also associated with a 2 to 4 fold risk of coronary artery disease.

Type 1 diabetes mellitus represents very low or unmeasurable plasma insulin with increased glucagon. Specifically, the immune response directed to β-cells induces type 1 diabetes because β-cells secrete insulin. A typical therapeutic plan for type 1 diabetes is to minimize hyperglycemia resulting from lack of natural insulin.

Fibroblast growth factor 21 (FGF21) is a novel metabolic modulator mainly produced by the liver that exhibits effective antidiabetic and lipid-lowering effects in animal models of obesity and type 2 diabetes mellitus. This hormone contributes to weight control and is involved in the response to malnutrition and ketoneogenesis in mice. The main sites of metabolism of FGF21 are adipose tissue, liver and pancreas. Experimental studies have shown improvement in diabetes compensation and dyslipidemia after FGF21 administration in diabetic mice and primates (Dostalova et al. 2009). FGF21 stimulates glucose uptake in mouse 3T3-L1 adipocytes with and without insulin and feeds and fasting blood glucose, triglyceride and glucagon concentrations in ob / ob and db / db mice and 8 week old olf ZDF rats in a dose dependent manner. Has been shown to provide a criterion for the use of FGF-21 as a therapy for treating diabetes and obesity (see, e.g., International Patent Application Publication No. WO03 / 011213).

Fibroblast growth factor (FGF) is a polypeptide that is widely expressed in developing and adult tissues. The FGF family usually consists of 22 members, FGF-1 to FGF-23. Members of the FGF family are highly conserved in both the genetic structure and the amino acid sequences between vertebrate species. There are 18 mammalian fibroblast growth factors (FGF1 to FGF10 and FGF16 to FGF23) that are grouped into six subfamily based on differences in sequence homology and phylogeny. Numbered “FGFs” that are not assigned to the subfamily—FGF homology factors (previously known as FGF11 to FGF14) —have high sequence identity with the FGF family, but do not activate the FGF receptor (FGFR) and are therefore generally FGF It is not considered to be a member of the family.

While most of FGFs act as local regulators of cell growth and differentiation, recent studies have suggested that FGF19 subfamily members, including FGF15 / 19, FGF21 and FGF23, exhibit significant metabolic effects by the endocrine mode. Members of the FGF19 subfamily regulate various physiological processes that are not affected by conventional FGF. Extensive metabolic activity of these endocrine factors includes phosphate, calcium and vitamin D homeostasis as well as regulation of bile acids, carbohydrates and lipid metabolism (Tomlinson et al. 2002, Holt et al. 2003, Shimada et al. 2004, Kharitonenkov et al. 2005, Inagaki et al. 2005, Lundasen et al. 2006).

FGF21 was originally isolated from mouse embryos. FGF21 mRNA was mostly abundantly expressed in the liver and to a lesser extent in the thymus (Nishimura et al. 2000). Human FGF21 is quite identical to mouse FGF21 (approximately 75% amino acid homology). Among the members of the human FGF family, FGF21 is most similar to FGF19 (about 35% amino acid homology) (Nishimura et al. 2000). FGF21 lacks the usual proliferative and oncogenic effects for most members of the FGF family (Kharitonenkov et al. 2005, Huang et al. 2006, Wente et al. 2006) (Ornitz and Itoh 2001, Nicholes et al) 2002, Eswarakumar et al. 2005).

Administration of FGF21 in obese leptin-deficient ob / ob and leptin receptor-deficient db / db mice and obese ZDF rats significantly reduced blood glucose and triglycerides, reduced fasting insulin levels, and reduced glucose clearance during oral glucose tolerance tests. clearance). FGF21 did not affect diabetes or food intake or body weight / composition in lean mice and rats over a two week course of administration. Importantly, FGF21 did not induce mitogenicity, hypoglycemia or weight gain at any dose tested when overexpressed in diabetic or healthy animals or in transgenic mice (Kharitonenkov et al. 2005). FGF21-overexpressed transgenic mice were resistant to diet-induced obesity.

Administration of FGF21 to rhesus macaques for 6 weeks reduced fasting plasma glucose, fructosamine, triglycerides, insulin and glucagon levels. Importantly, hypoglycemia was not observed during the study despite significant glucose-lowering effects. FGF21 administration also significantly lowered LDL-cholesterol, increased HDL-cholesterol, and slightly but significantly reduced weight, in contrast to mice (Kharitonenkov et al. 2005) (Kharitonenkov et al. 2007).

Further information can be obtained from the following references:

Enteric peptide Glucagon-like peptide-1 (GLP-1) is an incretin hormone, secreted in a nutrient-dependent manner. It stimulates glucose-dependent insulin secretion. GLP-1 also promotes β-cell proliferation and regulates glucose levels through the addition of glucose sensors, suppressing gastric emptying, food intake and glucagon secretion. Moreover, GLP-1 stimulates insulin secretion and reduces blood sugar in human subjects with type 2 diabetes. Exogenous administration of bioactive GLP-1, GLP-1 (7-27) or GLP-1 (7-36 amide) at doses that increase plasma concentrations approximately 3-4 times the physiological postprandial level is associated with type 2 diabetes. Fully normalize fasting hyperglycemia in patients (Nauck, MA et al. (1997) Exp Clin Endocrinol Diabetes, 105, 187-197). Human GLP-1 receptor (GLP-1R) is a 463 amino acid heptahelical G protein-coupled widely expressed in the pancreatic islet, kidney, lung, heart and multiple regions of the peripheral and central nervous system. Receptors. Within the islet, GLP-1R is overwhelmingly localized to islet β-cells. GLP-1R signaling action initiates a differentiation into a more endocrine-like phenotype, in particular the differentiation of precursors from human islets into functional β-cells (Drucker, DJ (2006) Cell Metabolism, 3 , 153-165).

Unfortunately, both FGF-21 and bioactive GLP-1, as well as other known drugs, have their own limited efficacy on the complex and multifactorial metabolic dysfunction that can be observed in type 2 diabetes or other metabolic disorders. This also applies to the efficacy in lowering blood glucose levels by the compounds themselves.

In accordance with the present invention, it has been surprisingly found that the combination of FGF-21 and GLP-1R agonists significantly lowers blood glucose levels to normo-glycaemic levels in a synergistic manner.

Accordingly, one aspect of the invention relates to pharmaceutical compositions containing at least one FGF-21 (fibroblast growth factor 21) compound and at least one GLP-1R (glucagon-like peptide-1 receptor) agonist.

"FGF-21 compound" refers to FGF-21 activity, in particular (i) native FGF-21, in particular human FGF-21, in particular human FGF-21 as set forth in SEQ ID NO: 1, or (ii) It is defined as a compound comprising an FGF-21 mimetic having FGF-21 activity.

"FGF-21 activity" is usually measured by FGF-21 activity tests generally known to those skilled in the art. The FGF-21 activity test is a "glucose uptake assay" as described, for example, in Kharitonenkov, A. et al. (2005), 115; 1627, No. 6). As an example of glucose uptake testing, adipocytes are starved for 3 hours in DMEM / 0.1% BSA, stimulated with FGF-21 for 24 hours, and KRP buffer (15 mM HEPES, pH 7.4, 118 mM NaCl, 4.8 mM KCl, 1.2). washing twice with mM MgSO ₄ , 1.3 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ , 0.1% BSA), followed by 2-deoxy-D- [ ¹⁴ C] glucose (2-DOG) (0.1 μCi, 100 μM) 100 μl of KRP buffer containing was added to each well. Control wells contained 100 μl of KRP buffer containing 2-DOG (0.1 μCi, 10 mM) to monitor for non-specificity. Absorption reactions are carried out at 37 ° C. for 1 hour, terminated by addition of Cytokalacin B (20 μM) and measured using a Wallac 1450 MicroBeta counter (PerkinElmer, USA).

Examples of FGF-21 mimetics include (a) a protein having at least about 96%, in particular 99% amino acid sequence identity, and FGF-21 activity with the amino acid sequence set forth in SEQ ID NO: 1, (b) a FGF-21 fusion protein or ( c) FGF-21 conjugates such as FGF-21 muteins, FGF-21-Fc fusion proteins, FGF-21-HSA fusion proteins or PEGylated FGF-21.

Examples of FGF-21 muteins are described, for example, in International Patent Application Publications WO2005 / 061712, WO2006 / 028595, WO2006 / 028714, WO2006 / 065582 or WO2008 / 121563. . Exemplary muteins are, for example, wild type human FGF-21, eg, human FGF-21 substituted at position 167 (serine), for example, the following substitutions: Ser167Ala, Ser167Glu, Ser167Asp, Ser167Asn, Compared to human FGF-21 with one of Ser167Gln, Ser167Gly, Ser167Val, Ser167His, Ser167Lys or Ser167Tyr, it is a mutein with reduced ability for O-glycosylation when expressed in yeast. Other examples include muteins exhibiting reduced deamidation compared to wild type human FGF-21, eg, substituted at position 121 (asparagine) of human FGF-21, for example Asn121Ala, Asn121Val, Asn121Ser, Asn121Asp Or mutein which is Asn121Glu. An alternative mutein is, for example, human FGF-21 having one or more non-naturally encoded amino acids as described generally in claims 29 of WO2008 / 121563. . Other muteins are, for example, polar but uncharged or charged amino acids, respectively, charged amino acids (eg, aspartate, glutamate) or polar but uncharged amino acids (eg, serine, threonine, asparagine). , Glutamine). Examples include Leu139Glu, Ala145Glu, Leu146Glu, Ile152Glu, Gln156Glu, Ser163Glu, Ile152Glu, Ser163Glu or Gln54Glu. Other muteins are, for example, human FGF-21, in particular Leu153 to amino acids selected from Gly, Ala, Val, Pro, Phe, Tyr, Trp, Ser, Thr, Asn, Asp, Gln, Glu, Cys or Met. It is a mutein that exhibits reduced susceptibility to proteolysis upon expression in yeast compared to human FGF-21 substituted by. Preferred FGF-21 muteins are mutated FGF-21 according to SEQ ID NO: 2 having a deletion of amino acids 1 to 28 of human FGF-21 (SEQ ID NO: 1) and containing additional glycine at the N-terminus.

Examples of FGF-21 fusion proteins are described, for example, in International Patent Application Publication Nos. WO2004 / 110472 or WO2005 / 113606 (eg, FGF-21-Fc fusion proteins or FGF-21-HAS). Fusion protein). "Fc" means the Fc portion of an immunoglobulin, eg, the Fc portion of IgG4. "HSA" means human serum albumin.

Examples of FGF-21 conjugates are described, for example, in International Patent Application Publications WO2005 / 091944, WO2006 / 050247, or WO2009 / 089396 (eg, glycol-linked FGF-21 compounds). . The glycol-linked FGF-21 compounds usually mount polyethylene glycol (PEG), for example, at cysteine or lysine amino acid residues or at introduced N-linked or O-linked glycosylation sites (herein referred to as "PEGylation"). FGF-21 "). The PEGylated FGF-21 compounds generally exhibit prolonged time action as compared to human FGF-21. Suitable PEGs have a molecular weight of about 20,000 to 40,000 daltons.

"GLP-1R agonist" is defined as a compound that binds to and activates a GLP-1 receptor, such as GLP-1 (glucagon-like peptide 1). The physiological action of GLP-1 and / or GLP-1R agonists is described, for example, in Nauck, MA et al. (1997) Exp. Clin. Endocrinol. Diabetes, 105, 187-195. have. In normal subjects, especially humans, these physiological actions may include, for example, glucose-dependent stimulation of insulin secretion, inhibition of glucagon secretion, stimulation of (pro) insulin biosynthesis, reduction of food intake, slowing gastric emptying and / or Includes ambiguous insulin sensitivity.

Suitable tests for discovering GLP-1R agonists are described, for example, in Thorkildsen, Chr. Et al. (2003), Journal of Pharmacology and Experimental Therapeutics, 307, 490-496; Knudsen, LB et al. (2007) ), PNAS, 104, 937-942, No. 3; Chen, D. et al. (2007), PNAS, 104, 943-948, No. 3; or US Patent Application US2006 / 0003417 A1 (e.g., See example 8). Briefly, in the "receptor binding assay", for example, purified membrane fractions of eukaryotic cells (eg, CHO, BHK or HEK293 cells) that harbor human recombinant GLP-1 receptors are, for example, Test compound or compound in the presence of human GLP-1, eg, ¹²⁵ G (eg, 80 kBq / pmol) human GLP-1 (eg, GLP-1 (7-36) amide) Incubate with them. Usually different concentrations of test compound or compounds are used and IC ₅₀ values are determined as concentrations that reduce the specific binding of human GLP-1. In a “receptor functional assay”, for example, a plasma membrane expressing a human GLP-1 receptor, eg, isolated from eukaryotic cells as described above, is prepared and incubated with the test compound. do. Functional testing is performed by measuring cAMP in response to stimulation by the test compound. In "receptor gene testing", for example, as described above, for example, expressing a human GLP-1 receptor and containing, for example, multiple response element / cAMP response element-driven luciferase reporter plasmids. The same eukaryotic cells are cultured in the presence of the test compound. cAMP response element-driven luciferase activity is measured as a response to stimulation by the test compound.

Suitable GLP-1R agonists are described, for example, in Drucker, DJ (2006) Cell Metabolism, 3, 153-165; Thorkildsen, Chr. (2003; supra); Chen, D. et al. (2007; supra) Knudsen, LB et al. (2007; supra); Liu, J. et al. (2007) Neurochem Int., 51, 361-369, No. 6-7; Christensen, M. et al. 2009), Drugs, 12, 503-513; bioactive GLP-1, as described in Maida A. et al. (2008) Endocrinology, 149, 5670-5678, No. 11 and US Patent Application US2006 / 0003417). GLP-1 homologues or GLP-1 substituents. Exemplary compounds include GLP-1 (7-37), GLP-1 (7-36) amide, Xtendine-4, liraglutide, CJC-1131, albugon, albiggluted, exenatide, exenatide -LAR, auxintomodulin, lixisenatide, geniproside, AVE-0010, short peptides with GLP-1R agonist activity and / or small organic compounds with GLP-1R agonist activity.

Specifically, human GLP-1 (7-37) has the amino acid sequence of SEQ ID NO. Human GLP-1 (7-36) amide has the amino acid sequence of SEQ ID NO: 4. Xtendine-4 has the amino acid sequence of SEQ ID NO: 5. Exenatides have the amino acid sequence of SEQ ID NO: 6, and auxintomodulin has the amino acid sequence of SEQ ID NO: 7. The amino acid sequence of lixisenatide is shown in SEQ ID NO: 8. The structure of the lixisenatide is exendin-4 (1-modified C-terminally by six additional lysine residues to resist immediate physiological degradation by DPP-4 (dipeptidyl peptidase-4). 39) is the basis. The amino acid sequence of AVE0010 is shown in SEQ ID NO: 9.

The chemical structure of liraglutide is shown in FIG. 1. Liraglutide was obtained by substitution of Lys 34 of GLP-1 (7-37) for Arg and by addition of C16 fatty acid at position 26 using the γ-glutamic acid spacer. The chemical name is [N-epsilon (gamma-L-glutamoyl (N-alpha-hexadecanoyl) -Lys ²⁶ , Arg ³⁴ -GLP-1 (7-37)].

The chemical structure of CJC-1131 is shown in FIG. 2. Albumin is bound to the C-terminus of GLP-1 by d-alanine substitution at position 8. CJC-1131 exhibits a very good combination of stability and bioactivity.

Other peptides with GLP-1R agonist activity are exemplarily described in US Patent Application Publication US2006 / 0003417, and small organic compounds with GLP-1R agonist activity are described in Chen et al. 2007, PNAS, 104, 943- 948, No. 3 or Knudsen et al., 2007, PNAS, 104, 937-942.

In a further aspect of the invention, the pharmaceutical composition additionally contains at least one anti-diabetic drug and / or at least one DPP-4 inhibitor.

Exemplary anti-diabetic drugs include,

a) insulin,

b) thiazolidinediones such as rosiglitazone or pioglitazone (see, eg, International Patent Application Publication No. WO2005 / 072769), metformin (N, N-dimethylimidodicarbonimidic-diamide) or

c) sulfonylureas [eg, chlorpropamide (4-chloro-N- (propylcarbamoyl) -benzenesulfonamide), tolazamide (N-[(azepane-1-ylamino) carbonyl ] -4-methyl-benzenesulfonamide), glyclazide (N- (hexahydrocyclopenta [c] pyrrol-2 (1H) -yl-carbamoyl) -4-methylbenzenesulfonamide) or glymepiride (3- Ethyl-4-methyl-N- (4- [N-((1r, 4r) -4-methylcyclohexylcarbamoyl) -sulfamoyl] phenethyl) -2-oxo-2,5-dihydro-1H- Pyrrole-1-carboxamide).

According to the present invention, "insulin" means amino acids exchange / deletion / addition as well as salts and combinations thereof, such as further modifications such as modified insulins and insulin analogues such as acylation or other chemical modifications. By naturally occurring insulin, modified insulin or insulin analogue, including combinations of insulin with. One example of this type of compound is insulin detemir, ie LysB29-tetradecanoyl / des (B30) human insulin. Another example may be insulin incorporating unnatural amino acids or commonly non-encoding amino acids (eg, D-amino acids) in eukaryotes (Geiger, R. et al., Hoppe Seylers Z. Physiol. Chem) (1976) 357, 1267-1270; Geiger, R. et al., Hoppe Seylers Z. Physiol. Chem. (1975), 356, 1635-1649, No. 10; Krail, G. et al., Hoppe Seylers Z. Physiol.Chem. (1971) 352, 1595-1598, No. 11). Another example is an insulin analogue in which the C-terminal carboxylic acid of either the A-chain or B-chain or both is substituted with an amide.

"Modified insulin" is preferably from acylated insulins having insulin activity, preferably acylated insulins at the B29 position, in particular at least one amino acid (s) of the A and / or B chains of insulin (Tsai, YJ et al. (1997) Journal of Pharmaceutical Sciences, 86, 1264-1268, No. 11). Other acetylated insulins are desB30 human insulin or B01 bovine insulin (Tsai, Y. J. et al., Supra). Other examples of acylated insulins are described, for example, in US Pat. No. 5,750,497 and US Pat. No. 6,011,007. An overview of the structure-activity relationship for modified insulin is provided in Mayer, J. P. et al. (2007) Biopolymers, 88, 687-713, No. 5. Modified insulins are usually prepared by chemical and / or enzymatic manipulation of insulin or a suitable insulin precursor (eg, preproinsulin, proinsulin or a truncated analog thereof).

"Insulin analogues" are preferably insulins with insulin activity, in particular one or more mutation (s), substitution (s), deletion (s) and / or addition (s), especially C- and in the A and / or B chains; Insulin with N-terminal truncation or extension, preferably des (B30) insulin, PheB1 insulin, B1-4 insulin, AspB28 human insulin (insulin aspart), LysB28 / ProB29 human insulin (insulin lispro), LysB03 / GluB29 Human insulin (insulin glulisine) or GlyA21 / ArgB31 / ArgB32 human insulin (insulin glargine). The only condition of an insulin analog is that it has sufficient insulin activity. Along with its discussion in which amino acid exchanges, deletions and / or additions are tolerated, an overview of the structure-activity relationship for insulin analogs is provided in Mayer, J. P. et al. (2007; supra). The insulin analogue is preferably one or more naturally occurring amino acid residues, preferably one, two or three of them substituted by another amino acid residue. Further examples of insulin analogs include C-terminal cleavage derivatives (eg des (B30) human insulin); B-chain N-terminal truncated insulin analogs (eg des PheB1 insulin or des B1-4 insulin); Insulin analogues in which the A- and / or B-chains have N-terminal extensions, including the so-called "pre-insulin" in which the B-chains have N-terminal extensions; And insulin analogues in which the A- and / or B-chains have C-terminal extensions. For example, one or two Arg can be added at the B1 position. Examples of insulin analogues are described in the following patents and their equivalents: US Pat. No. 5,618,913, European Patent Publications EP 0 254 516 A2 and EP 0 280 534 A2. An overview of insulin analogs in clinical use is provided in Mayer, J. P. et al. (2007; supra). Insulin analogs or their precursors are prepared using genetic engineering techniques well known to those skilled in the art, usually in bacteria or yeast, with subsequent enzymatic or synthetic manipulations as required. Alternatively, insulin analogues can be prepared chemically (Cao, Q. P. et al. (1986) Biol. Chem. Hoppe Seyler, 367, 135-140, No. 2). Examples of specific insulin analogs include insulin aspart (ie AspB28 human insulin); Insulin lispro (ie LysB28, ProB29 human insulin); Insulin glulisine (ie LysB03, GluB29 human insulin); And insulin glargine (ie, GlyA21, ArgB31, ArgB32 human insulin).

Exemplary DPP-4 Inhibitors

Citagliptin: (R) -4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro [1,2,4] triazolo [4,3-a] -pyrazine-7 (8H) -yl] -1- (2,4,5-trifluorophenyl) butan-2-amine,

Bildagliptin: (S) -1- [N- (3-hydroxy-1-adamantyl) glycyl] pyrrolidine-2-carbonitrile,

Saxagliptin: (1S, 3S, 5S) -2-[(2S) -2-amino-2- (3-hydroxy-1-adamantyl) -acetyl] -2-azabicyclo [3.1.0] Hexane-3-carbonitrile,

Linagliptin 8-[(3R) -3-aminopiperidin-1-yl] -7- (but-2-yn-1-yl) -3-methyl-1-[(4-methylquinazolin- 2-yl) methyl] -3,7-dihydro-1H-purin-2,6-dione) adogliptin (2-({6-[(3R) -3-aminopiperidin-1-yl] -3-methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl} methyl) -benzonitrile and

Berberines (eg Berberis, Goldenseal, Hydrastis canadensis and Coptis Kinensis), quaternary ammonium salts from isoquinoline alkaloid groups found in the roots, rhizomes, stems and bark of plants (Coptis chinensis).

Individual compounds of the pharmaceutical compositions of the present invention may be formulated in one formulation, or may be contained in several formulations, eg, for simultaneous or subsequent ie sequential administration (s) or combinations thereof.

According to the present invention, the combination of at least one FGF-21 compound and at least one GLP-1R agonist surprisingly produces a synergistic effect on lowering plasma glucose levels as shown by the animal model in the Examples. Animal models are ob / ob or obese mice and db / db mice. ob / ob mice are mutant mice that are unable to produce the hormone leptin that regulates appetite. As a result, ob / ob mice eat too much and become completely obese. It is a standard animal model for hyperglycemia, insulin resistance and obesity. Another standard animal model for diabetes is db / db mice with deficient leptin receptor activity. In addition, these mice are characterized by obesity, hyperglycemia and insulin resistance.

The pharmaceutical compositions of the present invention contain a therapeutically effective amount of an individual compound and a generally acceptable pharmaceutical carrier, diluent or excipient such as sterile water, physiological saline, bacteriostatic saline, ie about 0.9% mg / ml benzyl alcohol. Containing saline, phosphate-buffered saline, Hank's solution, Ringer-Lactate, lactose, dextrose, sucrose, trehalose, sorbitol, mannitol and the like. The composition is generally a solution or suspension. It may be administered orally, subcutaneously, intramuscularly, to the lungs, by inhalation and / or via sustained release administration. Preferably, the composition is administered subcutaneously.

The term “therapeutically effective amount” means an amount of a compound that produces the desired therapeutic and / or prophylactic effect without causing generally unacceptable side effects. Typical dosage ranges are from about 0.01 mg / day to about 1000 mg / day. The preferred dosage range for each therapeutically effective compound is about 0.1 mg / day to about 100 mg / day and the most preferred dosage range is about 1.0 mg / day to about 10 mg / day, in particular about 1-5 mg / day. .

For subsequent administration (s), the individual compounds of the pharmaceutical composition have a synergistic effect of the FGF-21 compound and the GLP-1R agonist, eg, in the "glucose tolerance test", as shown in the Examples, for example. It is administered for a period that can still be measured. The glucose tolerance test is a test that measures how quickly glucose is removed from the blood after administration of glucose. Glucose is most often given orally ("oral glucose tolerance test" or "OGTT"). The time period for the subsequent administration of the individual compounds, in particular the FGF-21 compound and the GLP-1R agonist, is usually within 1 hour, preferably within 30 minutes, most preferably within 15 minutes, in particular within 5 minutes.

In general, the application of the pharmaceutical composition to a patient is once or several times a day or once or several times over a longer period in which one week or even such cases can be tolerated. The most preferred application of the pharmaceutical composition of the present invention is subcutaneous application 1-3 times a day in combined doses.

The pharmaceutical compositions of the present invention lower blood sugar levels to normal-glycemic levels and increase energy expenditure by the use of faster and more efficient glucose, so that at least one metabolic syndrome and / or atherosclerosis, in particular type 1 or Useful for the treatment of type 2 diabetes, dyslipidemia, obesity and / or hyperlipidemia, especially type 2 diabetes.

Accordingly, the present invention also relates to the use of the above-described pharmaceutical composition (s) for the manufacture of a medicament for the treatment of at least one of the above mentioned diseases or disorders and to a method of treating at least one of the above mentioned diseases in a patient. It is about. The patient is particularly a type 1 diabetes patient, a type 2 diabetes patient, in particular a type 2 diabetes patient in a diet-treatment, a type 2 diabetes patient in a sulfonylurea-treatment, a far-advanced stage. ) Type 2 diabetes patients and / or type 2 diabetes patients undergoing long-term insulin treatment. A medicament may be prepared by methods known to those skilled in the art, for example by mixing a pharmaceutically effective amount of a compound or compounds with an acceptable pharmaceutical carrier, diluent or excipient, as described above.

The following figures and examples are for illustrative purposes only and are not intended to limit the invention.

1 shows the chemical structure of liraglutide.
2 shows the chemical structure of CJC-1131.
3 shows the results of an oral glucose tolerance test (OGTT) after 10 days subcutaneous injection of FGF-21 with AVE0010 in ob / ob mice.
4 shows plasma glucose levels over time after subcutaneous injection of FGF-21 with AVE0010 in ob / ob mice.
5 shows the results of OGTT after 21-day subcutaneous injection of FGF-21 with AVE0010 in db / db mice.
6 shows plasma glucose levels over time after subcutaneous injection of FGF-21 with AVE0010 in db / db mice.
Example
1. Treatment of ob / ob mice
Female ob / ob mice (B6.V-LEP OB / J, 6 weeks old) were obtained from Charles Rivers Laboratories (Sulzfeld, Germany). Mice were randomly assigned to treatment or vehicle groups and randomization was stratified by body weight and fed blood glucose levels. Animals were housed in 6 groups for 12h light-dark cycles at 23 ° C. All experimental methods were performed in accordance with the German Animal Protection Law.
ob / ob mice are vehicle ^{(PBS), 0.05㎎? ㎏ -1} ? day -1 AVE0010 ( SEQ ID NO: ^{9), 0.75㎎? ㎏ -1?} day -1 recombinant human FGF-21 (SEQ ID NO: 2), or the combined capacitance FGF-21 (SEQ ID NO: 2) and AVE0010 (SEQ ID NO: 9), (0.75 + 0.05 mg kg ^-1- day ^-1 ) were treated subcutaneously once a day. Mice were fed a standard rodent feed arbitrarily during the drug treatment period. Body weights were recorded daily and food intake was measured once a week throughout the study. One day before the first treatment and on day 10 of the study, blood glucose was measured by tail bleeding under feeding conditions. As shown in FIG. 4, blood glucose levels of treated mice became normal-glycemia. On day 8 of the study, a glucose tolerance test (OGTT) was performed. Fasting mice were orally administered 2 g · kg ⁻¹ glucose. Blood glucose was measured at the time point indicated by tail bleeding without anesthesia. The results of the OGTT are shown in FIG. 3. Compared to administration of FGF-21 alone or AVE0010 alone, glucose tolerance was markedly stronger by combination treatment. Combination treated obese animals were much more glucose resistant than lean control animals.
2. Treatment of db / db mice
The female db / db mice (BKS.Cg-m + / + Leprdb / J, 6 weeks old) is the vehicle ^{(PBS), 0.05㎎? ㎏ -1} ? Day -1 AVE0010, 0.75㎎? ㎏ -1? Day -1 recombinant Treatment with human FGF-21 (SEQ ID NO: 2) or a combined dose of FGF-21 (SEQ ID NO: 2) and AVE0010 (SEQ ID NO: 9) (0.75 + 0.05 mg-kg ^-1 -day ^-1 ) once daily did. Mice were fed at random. Blood glucose and HbA1c were measured under feeding conditions before 1 week and 4 weeks after the first treatment. After 21 days of treatment, the Oral Glucose Tolerance Test (OGTT) was initiated. Fasting mice were orally administered a 2 g · kg ⁻¹ glucose solution and blood glucose was measured at the indicated time points. The results are shown in FIGS. 5 and 6. Administration of the FGF21 + AVE0010 combination results in normalization of blood glucose and significantly improves glucose tolerance compared to vehicle treated obesity control. On the other hand, treatment with FGF21 or AVE0010 alone compared to the combination only leads to inhibition of blood glucose increase and a small improvement in glucose tolerance.

                         SEQUENCE LISTING <110> SANOFI   <120> PHARMACEUTICAL COMPOSITION FOR TREATING A METABOLIC SYNDROME <130> DE2010 / 012 <150> 10305070.4 <151> 2010-01-21 <160> 9 <170> Kopatentin 1.71 <210> 1 <211> 209 <212> PRT <213> Homo sapiens <400> 1 Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser 1 5 10 15 Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro             20 25 30 Asp Ser Ser Pro Leu Leu Gln Pro Gly Gly Gln Val Arg Gln Arg Tyr         35 40 45 Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg     50 55 60 Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu 65 70 75 80 Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val                 85 90 95 Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly             100 105 110 Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu         115 120 125 Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu     130 135 140 His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly 145 150 155 160 Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu                 165 170 175 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp             180 185 190 Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala         195 200 205 Ser      <210> 2 <211> 182 <212> PRT <213> unknown <220> <223> G linked to fragment of human FGF21 from H29 to S209 <400> 2 Gly His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln 1 5 10 15 Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala             20 25 30 His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln         35 40 45 Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile     50 55 60 Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp 65 70 75 80 Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe                 85 90 95 Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala             100 105 110 His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp         115 120 125 Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro     130 135 140 Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp 145 150 155 160 Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg                 165 170 175 Ser Pro Ser Tyr Ala Ser             180 <210> 3 <211> 31 <212> PRT <213> unknown <220> <223> Fragment of human GLP-1 (7 to 37) <220> <221> MOD_RES &Lt; 222 > (31) <223> AMIDATION <400> 3 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly             20 25 30 <210> 4 <211> 30 <212> PRT <213> unknown <220> <223> fragment of human GLP-1 (7-36) <220> <221> MOD_RES (222) (30) .. (30) <223> AMIDATION <400> 4 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg             20 25 30 <210> 5 <211> 39 <212> PRT <213> Heloderma suspectum <220> <221> MOD_RES (222) (39) .. (39) <223> AMIDATION <400> 5 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser             20 25 30 Ser Gly Ala Pro Pro Pro Ser         35 <210> 6 <211> 39 <212> PRT <213> Heloderma suspectum <220> <221> MOD_RES (222) (39) .. (39) <223> AMIDATION <400> 6 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Thr Leu Lys Asn Gly Gly Pro Ser             20 25 30 Ser Gly Ala Pro Pro Pro Ser         35 <210> 7 <211> 37 <212> PRT <213> Homo sapiens <220> <221> MOD_RES (222) (37) .. (37) <223> AMIDATION <400> 7 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn             20 25 30 Arg Asn Asn Ile Ala         35 <210> 8 <211> 44 <212> PRT <213> unknown <220> <223> GLP-1 like peptide <220> <221> MOD_RES (222) (44) .. (44) <400> 8 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser             20 25 30 Ser Gly Ala Pro Pro Ser Lys Lys Lys Lys Lys Lys         35 40 <210> 9 <211> 44 <212> PRT <213> unknown <220> <223> GLP-1 like peptide <220> <221> MOD_RES (222) (44) .. (44) <223> AMIDATION <400> 9 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser             20 25 30 Ser Gly Ala Pro Pro Ser Lys Lys Lys Lys Lys Lys         35 40

Claims (16)

A pharmaceutical composition containing at least one FGF-21 (fibroblast growth factor 21) compound and at least one GLP-1R (glucagon-like peptide-1 receptor) agonist. The pharmaceutical composition of claim 1, wherein the composition further comprises at least one anti-diabetic drug and / or at least one DPP-4 (dipeptidyl peptidase-4) inhibitor. The formulation of claim 1, wherein the FGF-21 compound (s), the GLP-1R agonist (s), optionally the anti-diabetic drug (s) and optionally the DPP-4 inhibitor are combined in one formulation. Or contained in several formulations. The method of claim 3, wherein the formulations of FGF-21 compound (s), GLP-1R agonist (s), optionally anti-diabetic drug (s) and optionally DPP-4 inhibitors, are suitable for simultaneous or subsequent administration (s). Pharmaceutical compositions. 5. The pharmaceutical composition of claim 1, wherein the FGF-21 compound is selected from FGF-21 or FGF-21 mimetic. 6. The protein, FGF-21 fusion protein, and / or FGF-21 conjugate of claim 5, wherein the FGF-21 mimetic has at least about 96% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and has FGF-21 activity The pharmaceutical composition is selected from. The pharmaceutical composition of claim 6, wherein the FGF-21 mimetic is selected from FGF-21 muteins, FGF-21-Fc fusion proteins, FGF-21-HSA fusion proteins, and / or PEGylated FGF-21. The pharmaceutical composition of claim 1, wherein the GLP-1R agonist is selected from bioactive GLP-1, GLP-1 homologs or GLP-1 substituents. The method of claim 8, wherein the GLP-1R agonist is GLP-1 (7-37), GLP-1 (7-36) amide, Xtendine-4, liraglutide, CJC-1131, albugon, albiggluted , Exenatide, exenatide-LAR, auxintomodulin, lexicinatide, geniproside, AVE-0010 (SEQ ID NO: 9), short peptides with GLP-1R agonist activity and / or GLP-1R agonists A pharmaceutical composition, selected from small organic compounds having activity. The pharmaceutical composition of claim 1, wherein the anti-diabetic drug is selected from metformin, thiazolidinedione, sulfonylurea, and / or insulin. The pharmaceutical composition of any one of claims 1 to 10, wherein the DPP-4 inhibitor is selected from cytagliptin, bilagliptin, saxagliptin, linagliptin, adogliptin and / or berberine. . The pharmaceutical composition according to any one of claims 1 to 11 for use in treating at least one metabolic syndrome and / or atherosclerosis. The pharmaceutical composition according to claim 12, wherein the metabolic syndrome is selected from diabetes mellitus, dyslipidemia, obesity and / or hyperlipidemia, especially type 2 diabetes. Use of a pharmaceutical composition as defined in any one of claims 1 to 11 for the manufacture of a medicament for the treatment of at least one metabolic syndrome and / or atherosclerosis in a patient. Use according to claim 14, wherein the metabolic syndrome is selected from diabetes mellitus, dyslipidemia, obesity and / or hyperlipidemia, in particular type 2 diabetes. The method according to claim 14 or 15, wherein the patient is a type 1 diabetes patient, a type 2 diabetes patient, in particular a type 2 diabetes patient in a diet-treatment, a type 2 diabetes in sulfonylurea-treatment The patient is selected from patients, far-advanced stage type 2 diabetes patients and / or type 2 diabetes patients undergoing long-term insulin treatment.

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